Many of the factors which participate in mammalian DNA replication are soluble in the sense that, during aqueous cell fractionation, they are covered in the soluble (high speed supernatant) cell fraction. In the absence of these factors, isolated nuclei or replicating chromosomes cannot join Okazaki pieces to longer strands of nascent DNA and cannot accomplish extensive movement of replication forks. When soluble factors are added back to isolated nuclei or chromosomes, ability to join Okazaki pieces and ability to move replication forks are restored. Previous attempts to isolate soluble factors have been hindered by (1) inadequate amounts of starting material, (2) poor recovery of activity, and (3) instability of recovered activity. Poor recovery of activity has been partly a consequence of the use of stimulation of overall DNA synthesis as an assay. This assay requires the cooperation of many factors in the correct proportions. We propose to fractionate soluble factors from 10 to the 12th power log-phase CV1-S cells. We shall assay each fraction for 14 different types of activity, ranging from stimulation of overall DNA synthesis by isolated cell nuclei to assays for DNases and proteases. Most of the assays will probe specific subreactions in the overall process of DNA replication (such as RNA priming, chain extension, gap-filling, and Okazaki piece joining) and thus will not depend on cooperation of so many factors as will overall replication. In any case, we shall take extraordinary precautions to (1) allow recombination of fractions in all possible ways and (2) stabilize all fractions. During the three years of this grant, we shall emphasize identifications of factors responsible for RNA priming, for synthesis of continuous strands, and for joining of Okazaki pieces. However, our combined experience in most aspects of DNA replication plus the broad approach we are taking should allow partial characterization of additional soluble factors.